The goal of this proposal is to answer fairly straightforward questions about how DNA damage and DNA binding proteins are effected by one another. Two systems have been selected for study, one involving interactions between transcription factors and their binding sites and another between nucleosome core proteins and DNA. For each system we will determine the distribution of UV damage in bound and free substrates using sequencing gel analysis of DNA incised at sites of UV photoproducts. We will test the hypothesis that, in addition to their direct inhibitory effects on transcription, UV photoproducts may effect gene expression through regulatory pathways as well. Initially, we will quantify the capacity of transcription factors to bind promoters in which either a cyclobutane pyrimidine dimer or (6-4) photoproduct has been positioned at a defined location. DNA substrates for these studies are purified using immunoseparation techniques. By using different types of pyrimidine dimers, the effects of varying degrees of helical distortion and unwinding on transcription factor binding can be ascertained. Should differences in transcription factor binding be observed in the UV-damaged substrates, we will use these substrates to determine the effects of promoter damage on the initiation and progression of transcription. Our second experimental approach is designed to determine the ability of core histones to reassemble on a damaged DNA template. Phased nucleosome substrates containing specific photoproducts at selected sites will be reconstituted in vitro. DNA protein interactions will be analyzed by EMSA, micrococcal nuclease digestion, and DNase I footprinting. The effect of alterations in nucleosome stability or positioning on gene expression will be tested using in vitro transcription. Data from these studies will increase our understanding of the role DNA damage and its effect on protein-DNA interactions play in regulating transcription.